Quantitative real-time polymerase chain reaction (qRT-PCR)

Quantitative Real-time polymerase chain reaction (qRT-PCR) is a method that can be used to amplify and quantify nucleic acids. The procedure follows the general principle of polymerase chain reaction. When combined with reverse transcription polymerase chain reaction , qRT-PCR can be used to monitor gene expression by quantifying mRNA levels. It is the most sensitive technique for mRNA detection and quantification currently available. (Kubista et al. 2006)

qRT-PCR is based on detection of a fluorescent signal produced proportionally during amplification of a PCR product. The fluorescence will increase as the amount of the PCR product increases and it is quantified after each completed PCR cycle. The cycle at

which the fluorescence exceeds detection threshold background fluorescence, a parameter known as the threshold cycle (CT) or crossing point (Cp), correlates to the number of target cDNA molecules present in the sample. During the first cycles the signal is weak and cannot be distinguished from the background. As the amount of product accumulates, a signal develops and increases exponentially. Eventually the signal levels off and saturates. The signal saturation is due to the reaction running out of some critical component. The amplification curves are separated in the growth phase of the reaction. This reflects the difference in their initial amounts of template molecules.

An example of amplification curve is presented in figure 5. (Nolan et al. 2006; Kubitsa et al. 2006)

Figure 5. q-RT-PCR amplification curve. Figure from (Kubista et al. 2006).

There are two types of quantification methods; relative quantification and absolute quantification. In the absolute quantification, the target concentration is expressed as an absolute value i.e. a copy number or concentration. In the relative quantification the target concentration is expressed in relation to the concentration of a house-keeping gene and a standard curve is used to obtain the concentrations of the target and the house-keeping genes. (Giuletti et al. 2001)

Figure 6. qRT-PCR standard curve. Amplification curves shown in logarithmic scale for five standard samples. The crossing points with threshold line are the Ct values.

In the inset the Ct values are plotted vs. the logarithm of the initial number of template copies in the standard samples. Figure from (Kubista et al. 2006).

For the absolute quantification of the sample templates a standard curve is constructed from standards of known concentration. These standards can be a purified PCR product or a purified plasmid that contains the target sequence. The CT values of the diluted standards are read out, and plotted versus the logarithm of the samples’ concentrations, number of template copies or dilution factor (figure 6.). The standard curve produces a linear relationship between CT and initial amounts of total RNA or cDNA, allowing the determination of the concentration of unknowns based on their CT values.

Relative quantification is the most commonly used technique. It is a mathematical model that calculates changes in gene expression as a relative fold difference between an experimental and calibrator sample. While this method includes a correction for nonideal amplification efficiencies, the amplification kinetics of the target gene and reference gene assays must be approximately equal because different efficiencies will generate errors when using this method. Consequently, a validation assay must be performed where serial dilutions are assayed for the target and reference gene and the

results plotted with the log input concentration for each dilution on the x-axis, and the difference in CT (target-reference) for each dilution on the y-axis. If the absolute value of the slope of the line is less than 0.1, the comparative CT method may be used. The PCR product size should be kept small (less than 150 bp) and the reaction rigorously optimized. (Technical Notes NO. LC 10/2003, Roche; Wong & Medrano, 2005)

When studying gene expression, the quantity of the target gene transcript needs to be normalized against the quantity of a reference gene transcript in the same sample.

Quantitative RT-PCR method requires correction for experimental variations due to differences in input RNA amount or in efficiencies of reverse transcription. The normalization is done by using housekeeping genes. An ideal housekeeping gene should be expressed at a constant level among different tissues of an organism, and should not be affected by experimental treatment itself. Because there is no gene that meets this criterion for every experimental condition, it is necessary to validate the expression stability of a control gene for the specific requirements of an experiment prior to its use for normalization. The various methods of normalization can be combined with different calculation methods, like the absolute standard curve method described previously. For example, when using relative quantification with external standards, a standard curve is used to obtain the concentration of the target and the reference gene.

(Giuletti et al. 2001)

All the real-time PCR systems detect a fluorescent dye. The two most commonly used formats for detecting fluorescence are independent assays and sequence-specific assays. The sequence-independent assay relies on fluorophores that bind to all double-stranded DNA molecules regardless of sequence (for example SYBR Green I).

Sequence-specific probe binding assays rely on fluorophores coupled to sequence-specific oligonucleotide hybridization probes, for example TaqMan-probes that only detect certain PCR products. (Technical Notes NO. 18/2004, Roche)

SYBR Green I binds to DNA double helix in a sequence-independent fashion. SYBR Green I barely fluoresces when it is free in solution, but its fluorescence emission is greatly enhanced when it binds to DNA, due to conformational changes in the dye.

When SYBR Green I binds to dsDNA minor groove, its fluorescence emission increases over 100-fold. During the various stages of PCR, the intensity of the fluorescence signal

will vary, depending on the amount of dsDNA that is present. Thus, the increase in SYBR Green I signal correlates with the amount of product amplified during PCR.

Since this assay detects both specific and non-specific PCR products, it must be carefully optimized and the product must be identified after the PCR run.

Figure 7. PCR reaction in the presence of SYBR Green I. Figure from (Technical Notes NO. 18/2004, Roche).

The PCR reaction with SYBR Green I is presented in figure 7. After denaturation, all DNA becomes single-stranded (Figure 7A). At this stage of the reaction, SYBR Green I dye will not bind and the fluorescence intensity is low. During annealing, the PCR primers hybridize to the target sequence creating small regions of double stranded DNA that SYBR Green I dye can bind, thereby leading to increased fluorescence (Figure 7B).

In the elongation phase of PCR, PCR primers are extended and more SYBR Green I dye can bind (Figure 7C). At the end of the elongation phase, the entire DNA is double-stranded and a maximum amount of dye is bound (Figure 7D).

Since SYBR Green I binds to any double stranded DNA, it cannot discriminate between different double stranded DNA species. This is why the specific product, nonspecific products and primer-dimers are detected equally well. However, a melting curve analysis is an appropriate tool to discriminate between product and primer-dimer and

should always be included in the SYBR Green I program. (Technical Notes NO.

18/2004, Roche)

Sequence-specific assays use probes labeled with fluorophores. These assays are highly specific because fluorescence increases only if the specific target is present in the reaction. Due to this sequence specificity, non-specific by-products, such as primer-dimers or will not be detected and the product identification by melting curve analysis is usually not required.

Single-labelled probes are a special type of simplified hybridization probe that can detect mutations and single nucleotide polymorphisms (SNPs). The so-called simple probe format requires only one hybridization probe, labelled with only one fluorophore, to achieve sequence specificity. Typically such a probe is designed to specifically hybridize to a target sequence that contains the SNP of interest. Once hybridized to its target sequence, the SimpleProbe probe emits more fluorescence than it does when it is not hybridized. As a result, changes in fluorescence are based solely on the hybridization status of the probe. (Technical Notes NO. 18/2004, Roche)

3 Aims of the research

The aim of this research was to study mRNA expression levels of HIF-1α and three hypoxia-inducible genes, CA9, CA12, and OPN, in NSCLC patients' plasma and in healthy controls. mRNA expression was investigated by qRT-PCR, and the normalization was done using two house-keeping genes; beta-2-microglobulin and ubiquitin C. The results were analysed statistically. The aim was also to evaluate whether the studied genes would have potential as clinical tumor markers.

4 Methods